The organization of the chromatin fiber within the nucleus may play an important role in the expression of eukaryotic genes, but little is known about the mechanisms regulating the establishment and maintenance of higher-order chromatin domains. Chromatin insulators appear to be important players in the establishment of these domains and they may regulate gene expression by controlling the organization of the DMA within the nucleus. The Su(Hw) insulator, originally found in the gypsy retrotransposon, is part of a family of insulators that use various DMA binding proteins to tether a common protein, CP190, to the chromosomes. Protein components of this insulator are present at several hundred sites on polytene chromosomes, but they concentrate at approximately 25 sites called insulator bodies located mostly in the nuclear periphery of diploid cells in interphase, causing the chromatin fiber to form rosette-like structures. Preliminary results suggest that the nuclear organization imposed by insulators changes during development. These changes may be regulated by covalent modification of insulator proteins. In this application, we propose to continue investigating the mechanisms controlling insulator function by analyzing in detail the role of ubiquitination and sumoylation of insulator proteins in the regulation of insulator activity. Amino acid residues critical for these modifications will be mutated and the activity and distribution of modified and non-modified proteins will be analyzed. Other subfamilies of the general CP190 insulator class will be studied in detail. Proteins responsible for DMA binding of these new insulator classes will be identified and the possibility that they are coordinately regulated by ubiquitination and sumoylation will be analyzed. The role of the nuclear lamina in the establishment and maintenance of insulator bodies will be determined. In particular, we will analyze the possibility that ubiquitination and/or sumoylation of lamin regulates the assembly of insulator bodies or their interaction with the nuclear lamina. Finally, we will examine the genome-wide distribution of active insulators in stem versus differentiated cells to determine whether the organization of the rosette structures formed by the coalescence of multiple insulators changes during development and cell differentiation. Results from these experiments will help in understanding how stem cells differentiate along various developmental pathways and how alterations in gene expression patterns may result in cancer. [unreadable] [unreadable] [unreadable]